WO2002016414A2 - Composition destinee a l'elimination des cellules b autoreactives - Google Patents

Composition destinee a l'elimination des cellules b autoreactives Download PDF

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WO2002016414A2
WO2002016414A2 PCT/EP2001/009714 EP0109714W WO0216414A2 WO 2002016414 A2 WO2002016414 A2 WO 2002016414A2 EP 0109714 W EP0109714 W EP 0109714W WO 0216414 A2 WO0216414 A2 WO 0216414A2
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cells
composition
mog
autoreactive
poly
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PCT/EP2001/009714
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WO2002016414A3 (fr
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Marcel Zocher
Patrick BÄUERLE
Torsten Dreier
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Micromet Ag
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Priority to EP01976136A priority Critical patent/EP1311549A2/fr
Priority to US10/362,591 priority patent/US7332168B2/en
Priority to AU2001295503A priority patent/AU2001295503A1/en
Publication of WO2002016414A2 publication Critical patent/WO2002016414A2/fr
Publication of WO2002016414A3 publication Critical patent/WO2002016414A3/fr

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/0005Vertebrate antigens
    • A61K39/0008Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503Immunoglobulin superfamily
    • C07K14/70532B7 molecules, e.g. CD80, CD86
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/30Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/30Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • composition for the elimination of autoreactive B-cells Composition for the elimination of autoreactive B-cells
  • the present invention relates to a composition for the selective elimination of autoreactive B-cells comprising at least one (poly)peptide construct consisting of at least two domains wherein one of said domains comprises an autoreactive antigen or (a) fragment(s) thereof specifically recognized by the Ig receptors of said autoreactive B-cells and wherein one of said domains comprises an effector molecule capable of interacting with and/or of activating NK-cells, T-cells, macrophages, monocytes and/or granulocytes.
  • said composition comprises at least one (poly)peptide construct consisting of at least two domains, wherein said domain comprising an autoreactive antigen or (a) fragment thereof is MOG or (a) fragment(s) thereof and wherein said domain comprising an effector molecule is an anti-CD3 receptor or an Fc-part of an immunoglobulin.
  • said domain comprising an autoreactive antigen or (a) fragment thereof is MOG or (a) fragment(s) thereof and wherein said domain comprising an effector molecule is an anti-CD3 receptor or an Fc-part of an immunoglobulin.
  • Described is- also the use of the afore-mentioned (poly)peptide construct for the preparation of a pharmaceutical composition for the treatment and/or prevention of an autoimmune disease.
  • the present invention relates to method for treating, ameliorating and/or preventing of an autoimmune disease.
  • Autoimmunity results from the failure of the immune system in tolerating self-reactive lymphocytes, resulting in an adaptive immune response against self antigens. When such immune responses are sustained, they cause lasting tissue damage and are classified as autoimmune diseases. Autoimmune diseases are generally divided into three types: B-cell dominant, T-cell dominant or combinational types. Pathogenic phenotypes of B-cell dominant autoimmune diseases are caused by circulating autoantibodies produced by autoreactive B-cells, while those of the T-cell dominant type are caused by direct tissue damage of activated T-cells against cells presenting autoreactive peptide- MHC complexes on their surface.
  • Antibody-mediated autoimmune diseases can be differentiated into two major groups based on their immunopathogenic mechanism. The first group comprises autoimmune responses against cell-surface or extracellular matrix antigens, while the second group consists of immune-complex diseases.
  • Examples of the first group of antibody-mediated autoimmune diseases are autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, myasthenia gravis, Goodpasture's syndrome, immunologically mediated blistering diseases like Pemphigus vulgaris and pemphigus foliaceus, and acute rheumatic fever.
  • Examples of the second group comprise mixed essential cryoglobulinemia, subacute bacterial endocarditis, and several rheumatic autoimmune diseases.
  • pemphigus vulgaris and pemphigus foliaceus are usually treated with glucocorticoids and, in some cases, also with immunosuppressive agents.
  • Myasthenia gravis treatment options include anticholinesterase medications, immunosuppressive agents, thymectomy, plasmapheresis or intravenous unspecific immunoglobulin.
  • Multiple sclerosis treatments include interferon beta, glucocorticoids, plasmapheresis.
  • pan-B cell antibody based approaches include immuno-toxins (WO 96/36360). Recently, an approach combining an autoantigen with an immunotoxin has been tested in vitro. The construct was a desmoglein3 (dsg3)-toxin fusion protein for the treatment of experimental pemphigus vulgaris (Proby, Brit. J. Dermatol. (2000) 142, 321-330). This approach, however, lacks efficacy. In Proby (2000), a cytotoxic effect of the dsg3-PE toxin was determined in vitro at concentrations of 50 microgram/ml.
  • dsg3-PE toxin was determined in vitro at concentrations of 50 microgram/ml.
  • the doses theoretically required for specific activity by dsg3-PE toxin are about 100 times higher than the MTD determined for PE toxin compounds. It is conceivable that further harmful toxicity could result from binding of the autoantigen- toxin construct to anti-dsg3 autoantibodies. The complex could then be bound and internalized by any kind of FcR-bearing immune cells, killing thereby non- autoreactive immune cells and leading to a general immunosuppression.
  • Approaches to treat multiple sclerosis include treatments which affect the overall immune system, like treatment with anti-inflammatory agents, comprising azathioprine, cyclophosphamide, prednisone, corticosteroids, cyclosporin A, calcineurin, rapamycin, beta-interferon ("Harrison's Principles of Internal Medicine", 14 th edition, McGraw-Hill publisher, 2415-2419; Wang, J. Immunol. 165 (2000), 548- 557).
  • a number of non-specific treatments are administered that may improve the quality of life including physical therapy and psycho-pharmacological agents.
  • Experimental approaches include peptide ligands to block T cell epitopes (Holz, J. Immunol.
  • Autoreactive B cells occur at a very low frequency. For example, in a frequency of about 10 "6 to 10 "7 of total B cells circulating in the blood of individuals suffering from multiple sclerosis. This low frequency has been a major impediment in the isolation of such cells from patients! Elimination of autoreactive B cells in vivo can therefore only be monitored indirectly via the determination of autoreactive antibody titers and is further complicated by the long half-life of antibodies in serum. Direct in vitro testing of the proposed concept for the elimination of autoreactive B cells using human ex-vivo samples from patients suffering from B cell mediated autoimmune disease has not been possible due to the low abundance of these cells.
  • the technical problem underlying the present invention was to develop and to provide for means and methods for preventing, treating and/or ameliorating antibody-mediated autoimmune diseases/disorders.
  • the present invention relates to a composition for the selective elimination of autoreactive B-cells comprising at least one (poly)peptide construct consisting of at least two domains wherein one of said domains comprises an autoreactive antigen or (a) fragment(s) thereof specifically recognized by the Ig receptors of said autoreactive B-cells and wherein one of said domains comprises an effector molecule capable of interacting with and/or of activating NK-cells, T-cells, macrophages, monocytes and/or granulocytes and/or capable of activating the complement system.
  • compositions as described herein are capable of selectively eliminating autoreactive B cells and thereby removing the pathological cell causing an autoimmune disorder/diseases.
  • autoreactive B-cells are described herein above and are known to the skilled person as, e.g., illustrated in Immunobiology, Janeway and Travers, 1996 by Current Biology Ltd/Garland Publishing Inc..
  • B lymphocytes bear on their surface highly diverse receptors each of which is specific for a particular antigen and which together are capable of recognizing a wide diversity of antigens.
  • the antigen receptor of B lymphocytes is a membrane-bound form of the antibody that these cells will secrete when activated, see, inter alia, p.
  • autoreactive B cells these antigens bound to the antigen receptors are self antigens. Autoimmune diseases are mediated by immune responses specific for self antigens; see p. 1 :18 in Immunbiology, Janeway and Travers, 1996 loc, cit.
  • autoreactive B-cells are B- cells of all differentiation states, resting or activated, which carry a B-cell Ig-receptor on this cell surface and which - due to this feature - are capable of binding to/interacting with (a) specific autoantigen(s).
  • B-cell specific markers are also known in the art and comprise the whole family of membrane bound Ig molecules, preferably lgM+, lgD+ or lgG+, preferably in combination with any of the following markers B220+ (CD45R+), CD19+, CD20+, CD22+, CD21+, CD38+, CD49c+, CD72, CD79 ⁇ , ⁇ +, CDw78+, MHC class II and CD43- (for resting B cells).
  • markers B220+ CD45R+
  • constructs comprising an autoreactive antigen or (a) fragment(s) thereof as described herein may be employed for the selective elimination of autoreactive B-cells and/or reduction of autoreactive immunoglobulins. This is surprising since the person skilled in the art would expect that the administration of autoantigen(s) and (a) fragment(s) thereof would lead to a more profound prevalence and/or disease state of autoimmune disorders.
  • composition in context of this invention, comprises at least one (poly) peptide construct as defined herein and/or at least one (poly) nucleotide comprising a nucleic acid molecule encoding for such one (poly) peptide construct.
  • Said composition optionally, further comprises other molecules, either alone or in combination, like e.g. molecules which are capable of modulating and/or interfering with the immune system.
  • the composition may be in solid, liquid or gaseous form and may be, inter alia, in a form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s).
  • said composition comprises at least two, preferably three, more preferably four, most preferably (poly)peptide constructs (and/or nucleic acid molecules encoding said constructs) as described in the invention.
  • selective elimination means elimination of the above mentioned autoreactive B-cells in vivo as well as in vitro. Said term also comprises ex vivo elimination, inter alia, by dialysis approaches. It is preferred that said selective elimination does not hinder the immunological response and/or only minimally influences the natural immunological defense. Preferably, said elimination does not compromise the T-cell mediated immune response and/or does not interfere with non-autoreactive B-cells.
  • said elimination is caused by cytolysis, most preferably said cytolysis is mediated by cytocidal cells, like, macrophages, monocytes, granulocytes, (cytolytic) T- lymphocytes, natural killer (NK) cells and/or lymphokine-activated killer (LAK) cells.
  • cytocidal cells like, macrophages, monocytes, granulocytes, (cytolytic) T- lymphocytes, natural killer (NK) cells and/or lymphokine-activated killer (LAK) cells.
  • At least one (poly)peptide construct as employed herein above relates to at least one (poly)peptide construct, at least two, at least three, at least four or at least five (poly)peptide constructs which may be comprised in the composition of the present invention.
  • At least two domains as used herein above comprises at least two domains, at least three domains, at least four domains and at least five domains in accordance with the invention.
  • autoreactive antigen or (a) fragment(s) thereof means antigens or (a) fragment(s) therof which are capable of elucidating and/or mediating an autoimmune response. Said fragment(s) thereof is/are preferably an epitope of said antigen.
  • said antigens and/or its fragment(s) comprise proteinaceous structures, yet, said autoreactive. antigen or (a) fragment(s) may also comprise, either alone or in addition to said proteinaceous structures, inter alia, carbohydrate moieties or lipids.
  • autoreactive antigen or (a) fragment(s) thereof is not limited to antigens occurring in and/or deriving from the subjects own body (autologous and/or endogenic antigens) but furthermore comprises foreign molecules which are capable of eliciting an autoimmune-response by binding and/or interacting with molecules peculiar to one's own body (for example via hapten-carrier complexes).
  • said term also comprises antigens, like microbial antigens/epitopes, that share properties, e.g. amino acid sequences, with mammalian molecules, e.g. proteins, and are capable of provoking an autoimmune- response.
  • composition of the present invention comprises a (poly)peptide construct comprising a domain with at least one autoreactive antigen or at least one fragment thereof.
  • said (poly)peptide construct comprises a domain comprising more than one autoreactive antigens and/or fragments and/or epitopes thereof.
  • Said domain comprising said autoreactive antigen or (a) fragment thereof may therefore comprise several autoantigens and/or fragment(s) thereof.
  • said domain comprises at least one, more preferred at least two, more preferred at least three, more preferred at least four and most preferred at least five autoreactive antigen(s) or (a) fragment(s).
  • effector molecule capable of interacting with and/or activating NK-cells, T- cells, macrophages, monocytes and/or granulocytes relates, in accordance with this invention, to molecules capable of engaging, inter alia, lymphocytes and/or Fc ⁇ R positive cells in effector mechanisms, like the cell-lysis and/or phagocytosis.
  • Said lymphocytes and/or Fc ⁇ R positive cells comprise the above mentioned NK-cells, macrophages, monophages, monocytes and/or granulocytes, as well as lymphokine- activated killer cells, neutrophiles, eosinophils.
  • effector molecule capable of activating the complement system relates to effector molecules which are capable of activating the classical as well as the alternative complement pathway. Furthermore, said term relates to effector molecules capable of activating any other form of complement mediated lysis.
  • effector molecules in accordance with the present invention are, inter alia, disclosed herein and exemplified in the appended examples.
  • the present invention relates to a composition wherein the above described (poly)peptide construct is a fusion (poly)peptide or a mosaic (poly)peptide.
  • Said fusion (poly)peptide may comprise merely the domains of the (poly)peptide construct as described herein above as well as several (a) functional fragment(s) thereof.
  • said . fusion poly)peptide comprises further domains and/or functional streches, Therefore, said fusion poly)peptide can comprise at least one further domain, said domain being linked by covalent or non-covalent bonds.
  • linkage (as well as the construction of the (poly)peptide constructs comprised in the composition of the present invention), can be based on genetic fusion according to the methods known in the art (Sambrook et al., loc. cit., Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989)) or can be performed by, e.g., chemical cross-linking as described in, e.g., WO 94/04686.
  • the additional domain present in the fusion poiy)peptide may preferably be linked by a flexible linker, advantageously a (poly)peptide linker, wherein said (poly)peptide linker preferably comprises plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of said further domain and the N- terminal end of the peptide, (poly)peptide or antibody or vice versa.
  • Said linker may, inter alia, be a Glycine, a Serine and/or a Glycine/Serine linker.
  • Additional linkers comprise oligomerization domains. Oligomerization domains facilitate the combination of two or several autoantigens or fragments thereof in one functional molecule.
  • Non-limiting examples of oligomerization domains comprise leucine zippers (like jun-fos, GCN4, E/EBP; Kostelny, J. Immunol. 148 (1992), 1547-1553; Zeng, Proc. Natl. Acad. Sci. USA 94 (1997), 3673-3678, Williams, Genes Dev. 5 (1991), 1553-1563;Suter, "Phage Display of Peptides and Proteins", Chapter 11 , (1996), Academic Press), antibody-derived oligomerization domains, like constant domains CH1 and CL (Mueller, FEBS Letters 422 (1998), 259-264) and/or tetramerization domains like GCN4-LI (Zerangue, Proc. Natl. Acad. Sci. USA 97 (2000), 3591-3595).
  • leucine zippers like jun-fos, GCN4, E/EBP; Kostelny, J. Immunol. 148 (1992), 1547
  • the (poly) peptide construct as described herein may comprise further domains, inter alia, domains which provide for purification means, like, e.g. histidine stretches.
  • immunomodulators comprise, but are not limited to cytokines, lymphokines, T cell co-stimulatory ligands, etc.
  • Adequate activation resulting in priming of naive T-cells is critical to primary immunoresponses and depends on two signals derived from professional APCs (antigen-presenting cells) like dendritic cells.
  • the first signal is antigen-specific and normally mediated by stimulation of the clonotypic T-cell antigen receptor that is induced by processed antigen presented in the context of MHC class-l or MHC class-ll molecules.
  • T-cell co-stimulatory ligand therefore denotes in the light of the present invention molecules, which are able to support priming of naive T-cells in combination with the primary stimulus and include, but are not limited to, members of the B7 family of proteins, including B7-1 (CD80) and 137-2 (CD86).
  • proteinaceous compounds providing the primary activation signal for T-cells can comprise, but are not limited to, anti-CD3-svFv fragments, anti-T-cell receptor svFv fragments or superantigens.
  • Superantigens directly bind to certain subfamilies of T-cell receptor variable regions in an MHC-independent manner thus mediating the primary T-cell activation signal.
  • the invention also relates to the effector molecule as defined herein, wherein the T-cell co-stimulatory ligand is a cell surface molecule or a fragment thereof expressed on antigen-presenting cells (APC).
  • the T-cell co-stimulatory ligand is a cell surface molecule or a fragment thereof expressed on antigen-presenting cells (APC).
  • the effector molecule as defined herein, binding to an APC may be a T-cell co-stimulatory factor like B7-1 (CD80) or B7-2 (CD86), or adhesion proteins like LFA-3 (CD58), ICAM-1 (CD54), ICAM-2 or ICAM-3 or like the CD137-ligand.
  • B7-1 CD80
  • B7-2 CD86
  • adhesion proteins like LFA-3 (CD58), ICAM-1 (CD54), ICAM-2 or ICAM-3 or like the CD137-ligand.
  • the effector molecule defined herein above may have receptor or ligand function, and may be an immuno-modulating " effector molecule or a fragment thereof.
  • An immuno-modulating effector molecule positively and/or negatively influences the humoral and/or cellular immune system, particulary its cellular and/or non-cellular components, its functions, and/or its interactions with other physiological systems.
  • Said immuno-modulating effector molecule may be selected from the group consisting of cytokines, chemokines, macrophage migration inhibitory factor (MIF; as described, inter alia, in Bernhagen (1998), Mol Med 76(3-4); 151-61 or Metz (1997), Adv Immunol 66,197-223) , T-cell receptors and soluble MHC molecules.
  • cytokines which are selected from the group consisting of interleukin(s), interferon(s), TNF(s) and VEGF (Veikkola Semin Cancer Biol 9(3), 211-20), wherein said interleukin(s) comprise, but are not limited to IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL— 8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-14, IL-15, IL-16, IL-17 and IL-18, wherein interferon(s) comprise IFN- ⁇ as well as IFN- ⁇ and IFN- ⁇ and wherein TNF(s) comprise members of the lymphotoxin superfamily like TNF- ⁇ and TNF- ⁇ (Gruss (1996) Int J Clin Lab Res 26(3),143-59).
  • interleukin(s) comprise, but are not limited to IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5
  • cytokines are well known in the art and comprise, inter alia, GM-CSF, G-CSF, M-CSF.
  • said immuno-modulating effector molecule is a chemokine and is selected from the group consisting of IL-8, Eotaxin, GRO ⁇ , GRO ⁇ , GRO ⁇ , IP-10, MCP-1 , MCP-2, MCP-3, MCP-4, MIG, MIP-1 ⁇ , MIP-1 ⁇ , NAP-2, RANTES, I309, Lymphotactin, SDF-1 and C5a.
  • Further effector molecules may be selected from the group of neuroprotective proteins, such as the neurotrophic growth factors.
  • neurotrophic growth factors examples include the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and/or the growth factors IGF-1 and bFGF, and/or the respective receptors of the aforementioned molecules (Connor B., Brain Res. Brain Res. Rev. 27 (1998), 1-39; Lewin, Annual Review of Neuroscience 19 (1996), 289-317; Tessarollo, Cytokine Growth Factor Rev. 9 (1998), 125-137; Snider, Cell 77 (1994), 627-638; Garcia-Estrada, Brain Res.
  • NGF neurotrophins nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • NT-3 neurotrophin-3
  • IGF-1 and bFGF the respective receptors of the aforementioned molecules
  • effector molecule comprising, inter alia, different immunomodulating effector molecules.
  • effector molecules are cytokines, like IL-2 and GM-CSF.
  • the (poly)peptide constructs comprised in the composition of the present invention may be constructs which comprise domains originating from one species, preferably from mammals, more preferably from human.
  • constructs which comprise domains originating from one species preferably from mammals, more preferably from human.
  • chimeric and/or humanized constructs are also envisaged and within the scope of the present invention.
  • the composition of the invention comprises a construct which is a cross-linked (poly)peptide construct.
  • said cross-linking may be based on methods known in the art which comprise recombinant as well as biochemical methods.
  • the present invention relates in a further embodiment to a composition as described herin above, wherein said autoreactive antigen or (a) fragment(s) thereof is selected from the group consisting of intracellular matrix proteins, extracellular matrix proteins, complement factors, nuclear antigens, cell surface receptors, nuclear receptors, lipids, soluble factors, membrane proteins, heat shock proteins, proteins with sequence similarity to microbial antigens, dietary components and proteins of intercellular structures.
  • said intracellular matrix protein is selected from the group consisting of keratin, filaggrin, antiperinuclear factor 7.
  • rheumatoid factor an IgM autoantibody directed against the Fc region of lgG2a
  • RF rheumatoid factor
  • RA rheumatoid arthritis
  • anti-A2/ RA33 antibodies Hassfeld (1989) Arthritis Rheum. 32: 1515-1520; Hassfeld (1993) Br. J. Rheumatol. 32: 199-203
  • antikeratin antibodies and the antiperinuclear factor 7 Youinou (1995) Int. Arch. Allergy Immunol. 107: 508-518; Sebbag (1995) J. Clin. Invest. 95: 2672-2679
  • anti-Sa antibodies Despres, N., G. Boire, F.J. Lopez-Longo, and H.A.
  • Anti-A2/RA33 autoantibodies are directed to the RNA binding region of the A2 protein of the heterogeneous nuclear rib ⁇ nucleoprotein complex (Skriner, J. Clin. Invest. 100 (1997), 127-135).
  • Antibodies to the Sa antigen cross-react in an example of molecular mimicry with a poorly soluble human protein that is present in normal tissues and that is distinct from all previously described RA-associated autoimmune systems (Depres, J. Rheumatol. 21 (1994), 1027-1033).
  • cytokeratin filament-aggregating protein filaggrin is the target of the so-called “antikeratin antibodies,” autoantibodies specific for rheumatoid arthritis (Simon (1993) J. Clin. Invest. 92,1387-93)
  • the present invention relates to a composition as described herein above, wherein said extracellular matrix protein is collagen. It is particularly preferred that said collagen is collagen type IV or collagen XVII. In this context, the autoreactive antigen or (a) fragment thereof may also be the non- collagenous domain of collagen.
  • Goodpasture's syndrome results from antibodies directed against collagen type IV, in particular the non-collagenous domain of the basement membrane collagen type IV (Butkowski et al. (1987) J. Biol. Chem. 262, 7874-7877; Saus et al. (1988) J.Biol.Chem. 263, 13374-13380).
  • the clinical manifestations of Goodpasture's syndrome are glomerulonephritis and pulmonary hemorrhage (Wilson, C, and Dixon, F. (1986) in the kidney (Berner, B., and Rector, F. eds) 3 rd Ed., pp800-889, W.B. Saunders Co., Philadelphia). Serum levels of autoantibodies to hemidesmosomal collagen XVII / BP180 were reported to correlate with disease activity in patients with bullous pemphigoid (see herein below).
  • the above mentioned complement factor is C5. It has been shown that the complement factor C5 is a potent autoantigen in rheumatoid arthritis (Volkman, J. Immunol. 158 (1997), 693-706; Grant, Cell Immunol. 167 (1996), 230-240)
  • the present invention relates to the above descibed composition wherein said nuclear antigen is selected from the group consisting of DNA, histones, snRNPs, topoisomerase I, ro (SS-A-Ro), la (SS- B-La), Scl-70, centromer protein (CENP), large ribonuclear particles (RNPs), AL Sm proteins, tRNA synthetase and Ku antigen.
  • said nuclear antigen is selected from the group consisting of DNA, histones, snRNPs, topoisomerase I, ro (SS-A-Ro), la (SS- B-La), Scl-70, centromer protein (CENP), large ribonuclear particles (RNPs), AL Sm proteins, tRNA synthetase and Ku antigen.
  • rheumatic autoimmune. diseases such as Systemic lupus erythematosus (SLE), progressive systemic sclerosis, polymyositis, mixed connective tissue disease (MCTD), or RA is the occurrence of autoantibodies to intracellular antigens (von Muehlen (1995) Semin. Erthritis Rhem. 24, 323-358; Peng (1997) in "Textbook of Rheumatology.” W.B. Saunders Company, Philadelphia, PA. 250-266).
  • these autoantibodies are often directed to components of large ribonucleoprotein (RNP) structures such as the ribosome or the spliceosome (van Venrooij (1995) Curr.
  • RNP ribonucleoprotein
  • SLE systemic lupus erythematosus
  • immune responses to SS-A 52-kDa and 60-kDa proteins and to SS-B 50- kDa protein have been shown in mothers of infants with neonatal lupus erythematosus (Yukiko (2000) Br. J. Dermatol. 142, 908-912).
  • Photosensitivity of lupus erythematosus was correlated with the expression of SS-A/Ro and SS-B/La antigens in skin biopsy specimens of patients (loannides (2000) Arch Dermatol 136, 340-346).
  • Wang et al. J. Clin. Invest. (1999) 104, 1265-1275
  • Circulating anticentromere CENP-A and CENP-B antibodies were identified in patients with diffuse and limited systemic sclerosis, systemic lupus erythematosus, and rheumatoid arthritis (Russo (2000) J Rheumatol.
  • Ku is a heterodimeric protein composed of approximately 70- and approximately 80- kDa subunits (Ku70 and Ku80) originally identified as an autoantigen recognized by the sera of patients with autoimmune diseases (Tuteja (2000) Crit Rev Biochem Mol Biol 35, 1-33).
  • the present invention relates furthermore to a composition as described herein wherein said autoreactive antigen or (a) fragment thereof is a cell surface receptor and wherein said a cell surface receptor is selected from the group consisting of TSH-receptor, Ach-receptor, asialo-glycoprotein receptor and platelet integrin Gpllb:llla.
  • Grave's disease is an autoimmune condition characterized typically by hyperthyroidism, thyroid hyperplasia, and additional signs of ophthalmopathy, pretibial myxedema, or acropachy.
  • the pathophysiological mechanisms responsible for thyrotoxicosis and thyroid hyperplasia are attributed ' to autoantibodies directed against the thyrotropin receptor (TSHr).
  • TSHr thyrotropin receptor
  • Said antibodies activate the TSH receptor, which results in cAMP-dependent stimulation of thyrocyte function and growth (McKenzien (1995) in "Endocrinology". ";. W.B. Saunders Co. Philadelphia, PA; pp 676-711 )
  • Autoantibodies directed against the acetylcholine receptor are involved in Myasthenia gravis, an autoimmune disease which leads to a reduction of the number of (Ach-R) at the muscular motor endplate (see, inter alia, Heitmiller (1999; Semin. Thorac Cardiovasc. Surg., 11, 41-46 or Atassi (1997)Crit. Rev. Immunol. ;17, 481-495).
  • autoantibodies directed against the human asialo glycoprotein receptor are described in autoimmune hepatitis (Bojic (1997) Med. Pregl. 50, 363-8)
  • the present invention provides for a composition wherein the soluble factor mentioned herein above is selected from the group consisting of l-antigen, Rh-blood group factor, 21-hydrolase enzyme, glutamic acid decarboxylase (GAD), insulin, (ICA) 512, lCAP-69, (tissue) transglutaminase (tTG), transaldolase, S100beta, oxidized low-density lipoprotein (ox-LDL), crystallin, CNPase, proteinase 3 and type I antigen.
  • Autoimmune hemolytic anemia is caused by antibodies against Rh blood group antigens and type I antigens, destroying red blood cells and resulting in anemia (Leddy (1993); J.
  • Celiac disease also refered to gluten sensitive enteropathy is characterized by IgA autoantibodies against anti-tissue transglutaminase and antiendomysial antibodies (EMA).
  • EMA antiendomysial antibodies
  • oxidized low-density lipoprotein oxidized low-density lipoprotein (ox-LDL) are thought to play a pivotal role in the pathogenesis of atherosclerosis and can serve as a marker of coronary artery disease in patients with familial hypercholesterolaemia (Paiker (2000) Ann Clin Biochem 37, 174-178)
  • IDDM Insulin-dependent diabetes mellitus
  • IDDM Insulin-dependent diabetes mellitus
  • autoreactive antibodies are present in patients with IDDM.
  • Contributions of the humoral response to the onset of disease and disease progression of IDDM was shown (Bonifacio (2000) Diabetes 49, 202- 208; Coleman (2000) Diabetologia 43, 203-209; Rulli, M. (1999) Autoimmunity 31 , 187-193).
  • Also a connection between autoantibodies to diabetes mellitus and celiac disease was shown (Galli-Tsinopoulou A (1999) Horm Res 52, 119-124).
  • ICA 512 is an autoantigen of insulin-dependent diabetes mellitus (IDDM) which is homologous to receptor-type protein tyrosine phosphatases (++PTPases).
  • IDDM insulin-dependent diabetes mellitus
  • ++PTPases receptor-type protein tyrosine phosphatases
  • ICA 512 is an intrinsic membrane protein of secretory granules expressed in insulin-producing pancreatic beta-cells as well as in virtually all other peptide-secreting endocrine cells and neurons containing neurosecretory granules (Solimena (1996) EMBO J 15, 2102-2014).
  • IDDM autoantigens in IDDM
  • ICAp69 Kerges (1996) Diabetes 45, 513-521).
  • Anti-insulin antibodies were shown to be linked to the onset of diabetes (Yu (2000) Proc Natl Acad Sci U S A 97, 1701-1706)
  • Granulomatosis also known Morbus Wegener (Hewis, Curr. Opin. Rheumatol. 12 (2000), 3-10) is caused by autoantibodies against proteinase 3, a constituent of neutrophil azurophilic granules.
  • the present invention provides for a composition as described herein above, wherein said heat shock protein is selected from the group consisting of alpha B-crystallin, Hsp27, HSP70 and HSP60.
  • Alpha B-crystallin and Hsp27 have been implied in multiple sclerosis (Agius, Acta Neurol. Scand. 100 (1999), 139-147).
  • HSP70 human myelin and to oligodendrocytes recognizing human autoantigens, including HSP70 and myelin protein CNP (Salvetti, J. Neuroimmunol. 64 (1996), 143-153; Salvetti, J. Autoimmun. 5 (1992), 691-702; Birnbaum, Ann. N.Y. Acad. Sci. 835 (1997), 157- 167; Jones, Immunol. Today 14 (1993), 115-118).
  • Antibodies against Escherichia coli and chlamydial Hsp60 were shown to crossreact with human HSP60 (Mayr, Circulation 99 (1999), 1560-1566).
  • Retinal autoantigens include Hsc70 (Ohguro, Invest. Ophthalmol. Vis. Sci. 40 (1999), 82-89).
  • the present invention relates, in a more preferred embodiment, to a composition as described herin above, wherein said membrane protein is selected from the group consisting of PLP, MAG, MBP, MOG, Golgi-proteins, cytochrome P450 (CYPs) and UDP-glucuronosyltransferase (UGTs).
  • said membrane protein is selected from the group consisting of PLP, MAG, MBP, MOG, Golgi-proteins, cytochrome P450 (CYPs) and UDP-glucuronosyltransferase (UGTs).
  • MS Multiple sclerosis
  • CNS central nervous system
  • oligodendrocytes a chronic inflammatory disease of the central nervous system (CNS) of autoimmune origin, characterized by focal demyelination, loss of oligodendrocytes, and astrocytic scar formation in advanced stages of the disease.
  • Histopathologically, acute inflammatory lesions are characterized by infiltrating lymphocytes and macrophages scattered throughout the periventricular white matter, spinal cord, brainstem and optic nerves. In the later stages of the disease, vascular infiltrates are less prominent, and loss of myelin and oligodendrocytes predominates.
  • MS has been widely classified to be a majorly T-cell mediated disease, it is now recognized that MS has both a T-cell and a B-cell component (Ewing (1998) Immunology and Cell Biology 76, 47-54; Weckerle (1999) Nature Medicine 5, 153- 154; Genain (1999) Nature Medicine, 5, 170-175; Schmidt (1999) loc.cit; Lindert (1999) Brain 122, 2089-2099).
  • myelin breakdown as the hallmark of the disease is brought about by the combined effects of autoantibodies against myelin proteins, complement activation, cytotoxic cells and cytokine-induced toxicity.
  • soluble proteins have been identified as being involved as autoantigens in multiple sclerosis, including the above discussed soluble proteins like, CNPase, transaldolase, S100 ⁇ or B-crystallin and/or other heat shock proteins
  • MBP myelin basic protein
  • MOG myelin oligodendrocyte glycoprotein
  • PLP PLP
  • MAG MAG
  • MOG protein a minor constituent of myelin sheaths (0.05% of total myelin) and exclusively expressed as a cell-surface protein on their outermost surface layer, has been shown to be the only single protein able to induce Chronic relapsing EAE (CREAE) in the Lewis Rat. CREAE is thought to be the most appropriate animal model for MS.
  • MOG is a membrane glycoprotein found predominantly in the outermost lamella of the myelin sheaths that wrap and insulate axons in the CNS.
  • MOG proteolipid protein
  • MOG peptides have been shown to bind specifically to disintegrating myelin around axons in lesions of acute multiple sclerosis and in MOG-induced EAE in the marmoset model for MS, providing direct evidence that autoantibodies against MOG mediate target membrane damage in central nervous system demyelinating disease (Genain (1999) Nature Medicine, 5, 170-175; Raine (1999), Ann. Neurol. 46, 144- 160).
  • MOG antibodies were shown to be common in patients with multiple sclerosis (Reindl (1999) Brain, 122, 2047-2056). Therefore, and due to the central role of MOG-specific B-cells in MS in a further embodiment of the present invention, the composition of the present invention is particularly useful for the selective elimination of these autoreactive lymphocytes (see herein below).
  • Cytochromes P450 and UDP-glucuronosyltransferases (UGTs) are targets of autoantibodies in several hepatic and extrahepatic autoimmune diseases (Obermayer-Straup (2000) Can.J Gastroenterol 14, 429-39) .
  • a number of Golgi proteins have been described as autoantigens, recognized by sera from patients with various forms of rheumatic diseases.
  • Golgi autoantigens are golgin-67, golgin-95/gm130, golgin-160, giantin, golgin-97, p230, golgin245 and p210 (Eystathioy, (2000) J Autoimmun 14, 179-187; Mancini,. (2000) J Cell Biol 149, 603-12, Linstedt (1993) Mol Biol Cell 4, 679-93; Griffith (1997) Arthritis Rheum 40, 1693-1702; Erlich (1996) J Biol Chem 271 , 18328-18337; Fritzler (1995) J Biol Chem 270, 31262-31268; Fritzler (1993) J Exp Med. 178, 49- 62; Fritzler (1984) J Immunol. 132, 2904-2908; Rios (1994) J Cell Biol 125, 997- 1013; Renier (1994) J Autoimmun 7, 133-143).
  • the present invention provides for a composition for the selective elimination of autoreactive B-cells as described herein above, wherein said proteins sharing sequence similarity with microbial antigens or dietary proteins/components are selected from the group consisting of antigens mimicking proteins, polypeptides and/or carbohydrate structures from Streptococcus, Klebsiella, Proteus, M. tuberculosis, adenovirus, poliovirus, measles virus, retrovirus, papilloma virus, gluten and/or butyrophilin.
  • Microbial antigens can share regions of amino acid sequences homology with mammalian proteins.
  • composition of the present invention is, inter alia, useful for treating, preventing and/or ameliorating autoimmune responses due to such an antigenic mimicry of microbial organisms.
  • exogenous antigenic mimicry are known in the art and, inter alia, described in Paul,
  • acute rheumatic fever is caused by antibodies against streptococcal cell-wall antigens that cross-react with cardiac muscle and lead to arthritis, myocarditis and late scarring of heart valves (Khanna (1997) J. Autoimmun. 10, 99-
  • antigenc mimicry which leads to autoimmune reactions, especially in rheumatoid arthritis, is the cross-reaction to HLA-DR antigens with proteins/(poly)peptides of Proteus mirabilis (see, eg. Ebringer (1992), Ann. Rheum.
  • hsp60 human heat shock protein 60
  • the measles virus P3 protein resembles the above described autoantigen
  • MBP and may elucidate EAE.
  • the present invention provides for a composition as described herein above, when said dietary component is gluten or butyrophilin.
  • dietary components are nutrients that share structural or sequence similarity with mammalian and/or human proteins or post- translational modifications of human proteins, like N- or O-linked glycans. Such dietary components can cause molecular mimicry and induce an autoimmune reaction. Examples of dietary components are gluten and the milk constituent butyrophilin. Celiac disease, also referred to as gluten sensitive enteropathy is characterized by IgA autoantibodies against anti-tissue transglutaminase and antiendomysial antibodies (EMA).
  • EMA antiendomysial antibodies
  • Butyprophilin shares sequence homology to MOG. Butyrophilin has been shown to modulate animal models of multiple sclerosis due to molecular mimicry with MOG (Stefferl, J Immunol 165 (2000), 2859-2865).
  • the present invention also relates to a composition wherein said protein of intercellular structures as described herein above is selected from the group consisting of desmoglein-1 (Dsy1), desmoglein-3 (Dsy3), desmocollin, desmoplakin, envoplakin, periplakin, BP180, BPAG-1 , BPAG-2 and HD1/plectin.
  • desmoglein-1 Dsy1
  • desmoglein-3 Dsy3
  • desmocollin desmoplakin
  • envoplakin envoplakin
  • periplakin BP180
  • BPAG-1 BPAG-2
  • HD1/plectin HD1/plectin
  • Pemphigus vulgaris and pemphigus foliaceus are caused by antibodies against keratinocyte adhesion molecules desmoglein 3 (Dsg3) and desmoglein I (Dsg1 ), respectively (Amagai, (1991) Cell 67, 869-877; Allen(1993), J Invest Dermatol 100, 685-91 ).
  • the present invention relates to a composition, as described herein wherein said T cells are cytotoxic T cells.
  • the present invention relates to a composition for the selective elimination of autoreactive B-cells comprising at least one (poly)peptide construct consisting of at least two domains wherein one of said domains comprises an autoreactive antigen or (a) fragment(s) thereof specifically recognized by the Ig receptors of said autoreactive B-cells and wherein one of said domains comprises an effector molecule capable of interacting with and/or of activating NK-cells, T-cells, macrophages, monocytes and/or granulocytes, wherein said effector molecule is a receptor-ligand or the Fc-part of an immunoglobulin. It is particularly preferred that said effector molecule specifically binds to a molecule of the CD3-receptor complex. It is even more preferred that said receptor-ligand is an antibody or (a) fragment(s) or derivative thereof or an aptamer.
  • antibody relates to monoclonal or polyclonal antibodies.
  • Polyclonal antibodies (antiserum) can be obtained according to conventional protocols.
  • Antibody fragments or derivatives comprise F(ab') 2 , Fab, Fv or scFv fragments; see, for example, Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press 1988, Cold Spring Harbor, NY,
  • the derivatives of the antibodies can be produced by peptidomimetics.
  • Nucleic acid aptamers are well known in the art and, inter alia, described in Famulok, Curr. Op. Chem. Biol. 2 (1998), 320-327.
  • the preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sites (Gold, Ann. Rev. Biochem. 64 (1995), 763-797).
  • Said " other receptors may, for example, be derived from said antibody etc. by peptidomimetics.
  • the above mentioned antibody derivative is a scFv directed against a molecule of the CD3 receptor complex.
  • the term "molecule of the CD3 receptor complex” comprises any invariable proteins and or fragments thereof, which comprise CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ (CD3 ⁇ and CD3 ⁇ are also known as TCR ⁇ and TCR ⁇ ). It is particularly preferred that said scFv is directed against the CD3 ⁇ chain of the T-cell receptor complex.
  • Single chain constructs comprising such a specificity are known in the art and, inter alia, described in Mack (1997), J. Immunol. 158, 3965-3970 or in the appended illustrative examples.
  • the present invention relates to a composition for the selective elimination of autoreactive B-cells comprising at least one (poly)peptide construct consisting of at least two domains wherein one of said domains comprises an autoreactive antigen or (a) fragment(s) thereof specifically recognized by the Ig receptors of said autoreactive B-cells and wherein one of said domains comprises an effector molecule capable of interacting with and/or of activating NK-cells, T-cells, macrophages, monocytes and/or granulocytes, wherein said domain comprising an autoreactive antigen or (a) fragment thereof is MOG or (a) fragment(s) thereof and wherein said domain comprising an immunological effector molecule is an anti-CD3 receptor or an Fc-part of an immunoglobulin.
  • a composition for the selective elimination of autoreactive B-cells comprising at least one (poly)peptide construct consisting of at least two domains wherein one of said domains comprises an autoreactive antigen or (a) fragment
  • compositions as disclosed herein are in particular useful for the elimination of autoreactive B-cells in vitro, in vivo and ex vivo.
  • the appended examples illustrate that not only in vitro depletions but also ex vivo as well as in vivo depletions are envisaged with the composition of the present invention.
  • the present invention also provides for compositions which may be employed for the reduction of auto-reactive, antigen-specific immunoglobulins.
  • MOG is one of the major autoantigens involved, in multiple sclerosis.
  • the protein and/or nucleotides sequence of MOG are known to the person skilled in the art and described, inter alia, in WO 95/06727 or US5532351.
  • Approaches to affect the T cell arm of multiple sclerosis include induction of tolerance using synthetic peptides against the activation of MOG-specific T-helper cells ( see, WO 95/07096, WO 96/12737, WO 97/35879, WO 99/12966). However, these approaches do not influence, ameliorate and/or modify the B-cell related symptoms of the autoimmune reaction in multiple sclerosis.
  • MOG belongs to the family of B7 homologous proteins, sharing the membrane topology and the extracellular immunoglobulin domain (Johns, (1999) J. Neurochem. 72, 1-9).
  • Several human homologues to MOG have been identified, but none of them has been reported to be an auto-antigen in MS.
  • One homologue to MOG has been described that is expressed in B lymphocytes (WO 98/33912).
  • fusion proteins of MOG or MOG homologues with Fc portions have been described (WO 98/33912, WO 99/23867), yet, said Fc-portion fusion proteins have been designed to enhance protein expression and for use as an affinity tag for purification. Often a protease site is introduced to remove the Fc portion after purification.
  • the present invention relates in a further embodiment to a composition for the selective elimination of autoreactive B-cells wherein said at least one (poly) peptide construct is encoded by (a) a polynucleotide comprising a nucleic acid molecule encoding the polypeptide as depicted in SEQ ID NO. 2 or 4; (b) a polynucleotide comprising a nucleic acid molecule having the DNA sequence as depicted in SEQ ID NO.
  • polynucleotide may be a naturally occurring nucleic acid molecule as well as a recombinant nucleic acid molecule.
  • Said polynucleotide/nucleic acid molecule may, therefore, be of natural origin, synthetic or semi-synthetic.
  • regulatory sequences may be added to the nucleic acid molecule of the invention.
  • promoters, transcriptional enhancers and/or sequences which allow for induced expression of the above described polynucleotide may be employed.
  • a suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. USA 89 (1992), 5547- 5551) and Gossen et al. (Trends Biotech. 12 (1994), 58-62).
  • polynucleotide/nucleic acid molecules may either be DNA or RNA or a hybrid thereof.
  • hybridizing in this context is understood as referring to conventional hybridization conditions, preferably such as hybridization in 50%formamide/6xSSC/0.1%SDS and 100 ⁇ g/ml ssDNA, in which temperatures for hybridization are above 37°C and temperatures for washing in 0.1xSSC/0.1 %SDS are above 55°C.
  • hybridizing refers to stringent hybridization conditions, for example such as described in Sambrook., “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989.
  • said polynucleotide/nucleic acid molecule may contain, for example, thioester bonds and/or nucleotide analogues. Said modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucleases in the cell. Said nucleic acid molecules may be transcribed by an appropriate vector containing a chimeric gene which allows for the transcription of said nucleic acid molecule in the cell.
  • the polynucleotide/nucleic acid molecule of the composition of the present invention may be a recombinantly produced chimeric nucleic acid molecule comprising any of the aforementioned nucleic acid molecules either alone or in combination.
  • the nucleic acid molecule of the invention is part of a vector.
  • (poly)peptide construct of the composition of the present invention is a (poly)peptide comprising the amino acid sequence as depicted in SEQ ID NO. 2 or 4.
  • (poly)peptide constructs are envisaged, wherein the sequence as shown in SEQ ID No.: 2 or 4 comprises at least one modification.
  • Said modification(s) may be selected from the group consisting of amino acid exchange(s), insertion(s), deletion(s), addition(s), substitution(s), inversion(s) and duplication(s).
  • Said modification(s) also comprise conservative and/or homologue amino acid exchange(s). For example, guidance concerning how to make phenotypically/functionally silent amino acid substitution is given in Bowie (1990), Science 247, 1306-1310.
  • variants of peptides of this invention include (i) substitutions with one or more of the non- conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature (poly)peptide construct with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the (poly)peptide construct with
  • the present invention also relates to peptides which are at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably 90%, more preferably at least 95% and most preferably 99% identical or homologous to the (poly)peptide construct as shown in SEQ ID NO: 2 or 4.
  • Specific strategies for obtaining (poly)peptide constructs described herein above are known in the art. These methods comprise recombinant and biochemical methods and are, inter alia, disclosed in Sambrook, loc. cit.. Said methods also comprise protein engineering or direct synthesis.
  • the present invention relates to a composition for the selective elimination of autoreactive B-cells comprising at least one polynucleotide encoding at least one (poly)peptide construct as described herein.
  • the embodiments described herein above may be applied, mutatis mutantis, for the polynucleotide/nucleic acid molecule encoding said at least one (poly)peptide construct. It is understood that compositions as described herein and comprising at least one polynucleotide encoding at least one (poly)peptide construct as described herein may also be employed for the selective reduction of autoreactive, antigen- specific immunoglobulins.
  • said polynucleotide/nucleic acid molecule is part of a vector.
  • said vector is an expression vector.
  • Said vector of the present invention may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering, and may comprise further genes such as" marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
  • Particularly preferred vectors are vectors as, inter alia, described in the appended examples and comprise, e.g. the expression vector CD19xCD3 pEF-dhfr.
  • the vector of the composition of the present invention may in addition to the polynucleotides/nucleic acid sequences described herein above, comprise expression control elements, allowing proper expression of the coding regions in suitable hosts.
  • control elements are known to the artisan and may include a promoter, a splice cassette, translation initiation codon, translation and insertion site for introducing an insert into the vector.
  • the nucleic acid molecule of the invention is operatively linked to said expression control sequences allowing expression in eukaryotic or prokaryotic cells. Control elements ensuring expression in eukaryotic and prokaryotic cells are well known to those skilled in the art.
  • regulatory elements ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript.
  • Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions.
  • Possible regulatory elements permitting expression in for example mammalian host cells comprise the CMV- HSV thymikine kinase promoter, SV40, RSV-promoter (Rous sarcome virus), human elongation factor l ⁇ -promoter, enhancers, like CMV enhancer or SV40- enhancer.
  • promoters including, for example, the tac-lac-promoter or the trp promoter, has been described.
  • elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide.
  • suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNAI , pcDNA3 (Invitrogene), pSPORTI , pEF-dhfr or prokaryotic expression vectors, such as lambda gt11, pDS or pET.
  • the vector may further comprise nucleic acid sequences encoding for secretion signals. Such sequences are well known to the person skilled in the art.
  • leader sequences capable of directing the peptides of the invention to a cellular compartment may be added to the coding sequence of the nucleic acid molecules of the invention and are well known in the art.
  • the leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a protein thereof, into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusionprotein including an C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • the vector of the composition of the present invention may also be an expression vector.
  • Gene therapy which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors, methods or gene-delivering systems for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res.
  • nucleic acid molecules and vectors described herein and comprised in the composition of the present invention may be designed for direct introduction or for introduction via liposomes, viral vectors (e.g. adenoviral, retroviral), electroporation, ballistic (e.g. gene gun) or other delivery systems into the cell. Additionally, a baculoviral system can be used as eukaryotic expression system for the nucleic acid molecules of the invention. As documented in the appended examples, (poly) peptide constructs comprised in the composition of the present invention may also be expressed in mammalian expression systems, for example in CHO-cells.
  • composition of the present nivention comprising nucleic acid molecule as described herin above and/or the above described vectors/hosts may be particularly useful in treating, preventing and/or ameliorating an autoimmune desease/disorder. Therefore, said compositions may be employed in gene therapy approaches.
  • said nucleic acids encoding the (poly)peptide constructs as described herein may be cloned into a gene delivering system, such as a virus
  • the of the present invention comprises a host transformed with the vector described herein above. It is even more preferred that said host is a mammalian cell, most preferred is a human cell.
  • the present invention relates to a composition as described herein above which further comprises a compound capable of selectively eliminating plasma cells and/or a compound capable of selectively eliminating (an) auto-antibody(ies).
  • said compound capable of selectively eliminating plasma cells is an antibody or (a) fragment(s) or a derivative thereof specifically detecting an plasma cell-specific epitope.
  • said compound capable of selectively eliminating (an) auto-antibody(ies) is an anti-idiotypic antibody or (a) fragment(s) or a derivative thereof specifically reacting with said auto- antibody(ies).
  • the present invention provides for compositions as described herein for the selective reduction of autoreactive immunoglobulins/for the selective elimination of auto-antibody(ies).
  • the constructs as disclosed herein and employed in the compositions of the present invention are not only capable of selectively eliminating autoreactive B-cells but also of reducing titers of autoreactive immunoglobulins.
  • said selective reduction of autoreactive immunoglobulins leads to a titer-reduction of at least 20%, at least 40%, at least 50%, at least 60%, most preferably at least 70%.
  • Titers of autoreactive immunoglobulins may be measured by methods known in the art and as shown in the appended examples, e.g.
  • the composition of the present invention is a pharmaceutical composition optionally comprising a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention may be particularly useful in preventing, ameliorating and/or treating autoimmune disorders/diseases, as described herein above and herein below.
  • Suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration.
  • compositions of the invention may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site, like the brain.
  • the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • Proteinaceous pharmaceutically active matter may be present in amounts between 1 ng and 10 mg per dose; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is a continuous infusion, it should also be in the range of 1 ⁇ g to 10 mg units per kilogram of body weight per minute, respectively. In context of the present invention, it is preferred that that the peptides of the present invention are employed in concentrations of less than 500 ⁇ g/ml, more preferred at less- than 100 ⁇ g/ml, more preferred of less than 10 ⁇ g/ml and most preferred of less than 1 ⁇ g/ml.
  • compositions of the invention may be administered locally or systemically.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the pharmaceutical composition of the invention may comprise further agents depending on the intended use of the pharmaceutical composition.
  • Said agents may be drugs acting on the immune system, like FK506, cyclosporin, IFNbeta, azathioprine, cyclophosphamide, prednisone, corticosferoids, cyclosporin A, calcineurin, rapamycin and neuroprotective agents such as the neurotrophins (NGF, BDNF, NT-3); see also Webster, Mult. Scler. 3 (1997), 113-120; Ebadi, Neurochem. Int. 30 (1997), 347-374.
  • compositions, also the pharmaceutical compositions, of the present invention may be tested for functionality by different approaches.
  • a surrogate test system several approaches can be chosen: 1 ) Establishment of a B-cell derived cell line expressing a membrane-bound Ig derived from a murine hybridoma cell line specific for the auto-antigen, 2) Hybridoma cells expressing membrane-bound Ig could be isolated using auto-antigen; 3) Alternatively, autoreactive B cells may be derived from transgenic mice with autoreactive B cells. Mouse models which may be employed are known in the art. For example, Litzenburger (see J. Exp. Med.
  • the above mentioned functionality approach comprises the measurement of circulating immunoglobulins and in particular of autoreactive immunoglobulins after administration of a composition as defined herein.
  • a measurement is shown in the appended examples and may easily be employed in samples from test animals, as well as in samples of humans.
  • said sample is a blood sample.
  • the present invention relates in another embodiment to the use of at least one (poly)peptide construct as defined herein, of at least one polynucleotide described herein above or encoding at least one (poly)peptide construct as defined herein or of at least one vector described herein above for the preparation of a pharmaceutical composition for the treatment, amelioration and/or prevention of an autoimmune disease, preferably of a human autoimmune disease.
  • Said pharmaceutical composition may be useful for the autoimmune diseases and disorders mentioned herein above and are particularly useful for the treatment, prevention or amelioration of diseases selected from the group consisting of Pemphigus vulgaris, Bullous pemphigoid, Goodpasture's syndrome, autoimmune haemolytic anemia (AIHA), rheumatoid arthritis, Systemic Lupus erythematosus, Grave's disease (autoimmune hyperthyroidism), contact dermatitis, M astenia gravis, juvenile diabetes, Sj ⁇ gren's syndrome, autoimmune throiditis, primary hypoadrenalism (Addison's disease), multiple sclerosis, thrombocytopenic purpura, pemphigous foliaceous, Morbus Wegener (granulomatosis) and celiac disease.
  • diseases selected from the group consisting of Pemphigus vulgaris, Bullous pemphigoid, Goodpasture's syndrome, autoimmune haemolytic anemia (AIHA), rheumatoi
  • the present invention also provides for a method of therapy, amelioration and/or prevention of an autoimmune disease comprising the administration to a subject in need of such therapy and/or prevention an effective amount of at least one (poly)peptide construct as defined herein,, of at least one polynucleotide as defined herein, of at least one vector defined herein or of at least one host of described herein
  • siad method be employed for the treatment and/or prevention of autoimmune disorders as described herein, like, but not limited to, Pemphigus vulgaris, Bullous pemphigoid, Goodpasture's syndrome, autoimmune haemolytic anemia (AIHA), rheumatoid arthritis, Systemic Lupus erythematosuas, Grave's disease (autoimmune hyperthyroidism), contact dermatitis, Myastenia gravis, juvenile diabetes, Sjogren's syndrome, autoimmune throiditis, primary hypoadrenalism (Addison's disease), multiple sclerosis thrombo
  • compositions in particular the pharmaceutical compositions, uses and methods of the invention can be used for all kinds of diseases hitherto unknown as being related to or dependent on auto-antigens and/or the production of auto-antibodies.
  • Said compositions, uses and methods of the invention may be desirably employed in humans, although animal treatment is also encompassed by the uses and methods described herein.
  • treatment in accordance with this invention, the terms "treatment”, “treating” and the like are used herein to generally mean obtaining a desired pharmacolocical and/or physiological effect. Said effect may be prophylactic in terms of completely or partially preventing a disease, in particular, an autoimmune disease, or a symptom thereof and/or may be therapeutic in terms of completely or partially curing a disease, in particular, an autoimmune disease, and/or (an) adverse effect(s) attributed to said disease.
  • treatment includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease.
  • Fig. 1 Overview of concept for the elimination of autoreactive B cells. Overview of elimination of autoreactive B cells using molecules comprising an autoantigen and an effector molecule domain. Abbreviations are ECR: Effector cell receptor; ECRbp: Effector cell receptor binding protein; Ag: Antigen.
  • Fig. 2 Schematic representation of autoantigen - effector domain molecules.
  • FIG. 3 Expression and purification of MOGxCD3.
  • MOGxCD3 was expressed in CHO cells and purified by cation exchange, followed by nickel chelate chromatography and gel filtration.
  • A 5 Gel filtration peaks are shown, separated by SDS-PAGE and analyzed by Coomassie-blue staining. Peak number 5 contains monomeric MOGxCD3 protein of a molecular weight of approximately 47kDa.
  • B Gel filtration peaks were separated on SDS-PAGE identical to A, transferred to a nitrocellulose membrane and incubated with an anti-MOG antibody (8.18-C5). Only peak 5 containing the 47kDa monomeric MOGxCD3 reacts with monoclonal anti- MOG antibody (8.18-C5). Higher molecular weight bands present in peaks 1 to 4 are not recognized by anti-MOG antibody.
  • MOG-Fc fusion protein was expressed in CHO cells and " purified by affinity chromatography using Protein A sepharose. Purified protein was separated by SDS-PAGE and analyzed by coomassie-blue staining (A) in the presence of the reducing agent DTT, or by Western blotting on nitrocellulose (B), followed by detection using a monoclonal antibody against MOG (8.18-C5). As expected, MOG-Fc fusion protein has a molecular weight of approximately 50kDa under reducing conditions (+DTT), but runs at a molecular weight of approximately 115kDa in the absence of DTT (-DTT), indicative for a dimer under non-reducing conditions. Detection in (B) was with an anti-Fc monoclonal antibody.
  • Fig. 5 Detection of MOG fusion protein in ELISA. ELISA plates were coated with anti-MOG antibody 8.18-C5 to capture the fusion proteins by their MOG domain.
  • A Bound MOG-Fc fusion protein was detected with an AP-conjugated anti-human Fc antibody.
  • MOGxCD3 fusion protein was detected by an AP-labeled chicken versus singlechain Fv anti-CD3 polyclonal antibody.
  • FIG. 7 Binding of MOGxCD3 to human T-cells.
  • MOGxCD3 was incubated with human CD3-positive PBMCs, and bound fusion protein was detected with monoclonal anti-MOG antibody followed by FITC-labeled anti-mouse lgG1 antibody . Labeled cells were subjected to FACS analysis.
  • Fig. 8.18-C5 hybridoma cells show surface expression of lgG1.
  • Hybridomas were cultivated in serum-free medium for 4-5 months, and cell-surface lgG1 expression was examined by FACS analysis (A) with FITC-labelled anti-lgG1 antibody and (B) with biotinylated rMOG (recombinant MOG extracellular domain), followed by FITC-labeled streptavidin.
  • Fig. 9 Cytotoxic activity of autoantigen-effector proteins. FACS-based cytotoxicity assay with MOGxCD3 fusion protein. 8.18-C5 target cells were incubated with human CD3+ PBMCs and serial dilutions of MOGxCD3 protein in RPMI/10% FCS. Incubation was performed for 16h at 37°C/5% C0 2. After incubation, target cells were labeled with anti-lgG1 antibody and analyzed for viability through propidium iodide staining. Viable murine lgG1(+) cells were counted and expressed as percentage of viable target cells in control: (A) M0GxCD3, (B) MOG-Fc fusion protein.
  • Fig. 10 Specificity of elimination of autoreactive B cells. MOGxCD3 cytotoxicities. Parameters shown represent: specific toxicity as described in Fig 9; to measure spontaneous lysis target and effector cells had been incubated separately for the duration of the assay and were combined just prior to analysis: (A) MOGxCD3, (B) MOG-Fc.
  • Fig. 11 Cytotoxicity of MOGxCD3 is inhibited by recombinant MOG.
  • MOGxCD3 was used at 0.1 ⁇ g/ml in FACS-based cytotoxicity assay as described for Fig. 9.
  • Recombinant, biotinylated MOG was added to obtain a final concentration of 20 ⁇ g/ml.
  • MOGxCD3 binds to 8.18-C5 target cells. Binding was assessed with FITC-labeled anti-HIS antibody (Dianova) at 10 ⁇ g/ml.
  • Cytotoxic activity of MOG x CD3 is dependent on effector cell to target ratio. Assay was performed as described for Fig. 9 with varying concentrations of effector cells and a constant number of 8.18-C5 target cells.
  • Fig. 14 Flow cytometric analysis of cell binding properties of MOG-Fc.
  • the monocyte/macrophage cell line p388.D1 expressing murine Fc receptors was incubated with MOG-Fc. Bound MOG-Fc was detected via FITC-labeled anti-human Fc ⁇ antibody by flow cytometry (left panel). Binding was quantitated and expressed as mean fluorescence intensity (MFI) (right panel).
  • target cells 8.18-C5 target cells were incubated with human PBMCs and 10 ⁇ g/ml MOG-Fc protein in RPMI/10% FCS. Incubation was performed for 16 h at 37°C and 5% C0 2 . After incubation, target cells were labeled with anti-murine lgG1 antibody (mlgG) and analyzed for viability through propidium iodide (PI) staining.
  • PI propidium iodide
  • Column 1 incubation of PBMCs and target cells in the absence of isotype control and MOG-Fc.
  • Column 2 the effect of a recombinant human lgG1 (isotype control).
  • Column 3 - the effect of MOG-Fc under identical assay conditions.
  • Mouse B cell line TIB-208 is expressing cell surface lgG1 of non-MOG specificity.
  • Column 1 unspecific lysis of TIB-208 cells in the presence of human PBMC.
  • Column 2 the effect of MOG-Fc (10 ⁇ g/ml) on the viability of TIB-208 cells.
  • Column 3 the effect of MOG-Fc (10 ⁇ g/ml) on hybridoma line 8.18-C5 under identical assay conditions. Error bars indicate S.D. values of triplicates.
  • Single cell suspensions of spleens from anti-MOG transgenic mice were prepared. Splenocytes were cultured in the absence (dark columns) or presence (white columns) of MOG-Fc (10 ⁇ g/ml). After incubation, the B cell population was analyzed by flow cytometry using antibodies against the B cell markers IgM, IgD and CD19. The frequency of cell populations is expressed as percentage of total live cells within the lymphocyte gate. Error bars represent S.D. values of triplicates.
  • Anti-MOG transgenic mice were left untreated (control) or treated i.p. with 100 ⁇ g of MOG-Fc protein on days 1 and 3.
  • PBS PBS
  • peripheral blood was collected by tail bleeding and analyzed for anti-MOG reactive B cells via FACS staining using recombinant MOG-Fc and PE-conjugated anti-human Fc antibody (ICN).
  • B cells derived from anti-MOG transgenic mice with BL/6 background were transferred intraveniously into wildtype BL/6.
  • Peripheral blood was collected by tail bleeding 24 h (Fig. 19A) and 72 h (Fig. 19B) post-treatment and serum was analyzed for anti-MOG specific IgG titers by ELISA.
  • Example 1 Expression vectors for autoantigen fragment - ⁇ CD3 and -Fc fusion proteins
  • Expression of the construct of interest is driven by the promotor of the human elongation factor alpha (Kufer; PNAS 92 (1995): 7021).
  • This promotor is known to be very efficient in virtually all eukaryotic cells, thereby making this expression system a powerful tool for high protein expression without limitations regarding the selected eukaryotic host cell line.
  • a versatile multiple cloning site (MCS) facilitates the cloning of the construct.
  • the expression of the construct of interest is linked to the expression of the selection marker dihydrofolate reductase (DHFR) via the internal ribosomal entry site (IRES). This arrangement assures that almost all stably transfected cells will express the construct, as both genes depend on the promotor of EF ⁇ .
  • a strong polyadenylation signal for both genes is provided by the SV40 polyadenylation site, and the pUC18 backbone of the plasmid provides a well- characterized plasmid backbone with ampicillin resistance for bacterial selection.
  • Example 2 Construction of an exemplified antigenxeffector moleculejAn autoantigen x ⁇ CD3 fusion protein: MOGxCD3
  • the expression vector pEF-dhfr CD19xCD3 (Loffler; Blood 95 (2000): 2098) was used as the basis for the construction of MOGxCD3 by removing the region coding for the single-chain ⁇ CD19 antibody. As described below, the remaining ⁇ CD3 expression cassette was used for insertion of cDNA coding for the auto-antigenic domain of the human MOG protein and provides an ideal system for further cloning of auto-antigen x ⁇ CD3 fusion proteins (Fig. 2A).
  • RT Superscript II Reverse Transcriptase
  • Primer 2 (MOG-Ex 3'): 5'-CATCCGGATCCAGGGCTCACCCAGTAGA-3' (Seq ID No 6)
  • Primers were designed to amplify the first 462 bases of the coding region for the leader sequence and extracellular domain of the human MOG protein.
  • the primers contained EcoRI and BspEI restriction sites at 5 ! and 3' ends, respectively.
  • Polymerase chain reactions (PCR) conditions were: 50 pmol primer, 1 ⁇ l dNTPs 10mM, 4 ⁇ l cDNA, 5 ⁇ l Pfu-buffer 5x (Stratagene) and 5 U Pfu-Polymerase (Stratagene) in a final volume of 50 ⁇ l.
  • the final product of 474bp was verified on a ethidium bromide stained 2% agarose gel.
  • the expression vector CD19xCD3 pEF-dhfr was digested with EcoRI and BspEI, leading to removal of the fragment coding for the anti-CD19 scFv domain.
  • the remaining vector-anti CD3 scFv portion was gel-extracted (gel extraction kit, Qiagen). Equally, MOG-ex was partially digested with BsaWI and EcoRI, and the corresponding fragment of 474bp of length isolated via gel-extraction. BsaWI restriction was chosen due to this enzyme's insensitivity to dam-methylation at the original BspEI restriction site. DNA was eluted in 30 ⁇ l Tris pH 8.5 and stored at - 20°C.
  • RT Superscript II Reverse Transcriptase
  • Primer 3 (lgG1-Fc 5'): 5'-TATCCGGAGAGCCCACCTCTTGTGACAAAAC-3' (Seq ID No 7)
  • Primer 4 (lgG1-Fc 3'): 5'-GTGTCGACTCATTTACCCGGAGACAGGG-3' (Seq ID No 8) .
  • an even more preferred primer is the following:
  • Primers were designed to amplifythe 699 bases coding for the Fc part of the human lgG1 backbone, while introducing BspEI and Sail restriction sites at 5' and 3' ends of the amplified fragment, respectively. Amplification was performed according to standard PCR protocols. Briefly, 50 pmol/ each of appropriate primer, 1 ⁇ l dNTPs 10mM, 4 ⁇ l cDNA, 5 ⁇ l Pfu-buffer 5x (Stratagene) and 5 U Pfu-Polymerase (Stratagene) were added to a final volume of 50 ⁇ l in H20. The final product, containing the flanking restriction sites, was 711 bp in length.
  • DNA was recovered from PCR reaction mixture according to the manufacturer's suggestions (Boehringer High Pure PCR Product Purification Kit, cat. no. 1 732 676). Blunt-ended PCR products generated by Pfu DNA polymerase were ligated into pCR-script vector (Stratagene #211188) according to manufacturer's protocol (Stratagene). Plasmids were transformed into competent E. coli strain XL-1 Blue using 4 ⁇ l of ligation product added to 50 ⁇ l of E. coli. The mixture was incubated on ice for 10 min., 1 min. at 42°C, and then again on ice for 2 min.
  • expression vector CD19xCD3 pEF- dhfr was subjected to restriction with EcoRI and Sail, leading to removal of the fragment coding for CD19xCD3.
  • the remaining linearized vector was gel-extracted (gel extraction kit, Qiagen).
  • MOG-ex was partially digested with BsaWI and EcoRI, and the corresponding fragment of 474bp of length was isolated via gel- extraction as described above.
  • BsaWI restriction was chosen due to this enzyme's insensitivity to dam-methylation at the original BspEI restriction site.
  • DNA was eluted in 30 ⁇ l Tris pH 8.5 and stored at -20°C.
  • Ligation of isolated DNA fragments was performed with equal volumes of extracted DNA and 5 U of T4 DNA Ligase in a total volume of 20 ⁇ l in 1x T4 buffer (Roche Biochemicals). Ligation was allowed to proceed for 30 min. at room temperature (RT). Of each ligation reaction, 3 ⁇ l were .used to transform E. coli XL-1 Blue as described above. Colonies were picked and subjected to MiniPrep analysis. Following analytical restriction enzyme digestion, appropriate clones were sequenced (Sequiserve, Kunststoff). Correct clones were grown in 300 ml LB-Amp medium, and plasmid DNA was isolated using the Qiagen plasmid prep kit as described above.
  • CHO cells were plated at 3*10 5 / well in tissue culture 6-well plates and incubated at 37°C overnight. 3 ⁇ g of DNA were pipetted in sterile Eppendorf tubes, supplemented with 100 ⁇ l MEM- ⁇ medium (Gibco BRL) and 10 ⁇ l SuperFect transfection reagent (Qiagen) and incubated for 10 min. at RT. 600 ⁇ l of MEM- ⁇ medium were added, and the. reaction mixture was transferred to CHO cells. Following a 2h-incubation at 37°C, the supernatant was aspirated, cells were washed once with PBS, and 2 ml MEM- ⁇ medium (10% FCS, HT-supplement 1:100) were added to each well.
  • Transfection efficiency was determined to be 10% via standard ⁇ -galactosidase control transfection.
  • transfected cells were transferred to 10 ml cell culture bottles (Nunclone ⁇ , Nalge Nunc International) and selected for expression of the dhfr vector via growth in non-supplemented MEM- ⁇ medium plus 10% dialysed FCS.
  • transfectants were further selected by addition of 20 nM methotrexate (MTX) to the selection medium. Cells were passaged 3 times, whereafter MTX concentration was increased to 100 nM. Following a further 3 passages, MTX was added to a final concentration of 500 nM.
  • MTX methotrexate
  • Stably transfected CHO-cells were transferred to 500 ml roller-bottles (Nalge Nunc International) in 250 ml MEM- ⁇ , 500 nM MTX and 5% dialysed FCS. The following day, another volume of medium was added without FCS to obtain a final concentration of 2.5% FCS. Cells were grown for 1 day post confluency. Cells were separated from the supernatant by centrifugation at 4500 rpm, 30 min. in a Rotanta 46 centrifuge, and recombinant protein was purified from cell culture supernatant in a three-step purification process including cation exchange chromatography, immobilized metal affinity chromatography (IMAC) and gel filtration. GradiFrac System (Pharmacia) was used for chromatography. All chemicals were of research grade and purchased from Sigma (Deisenhofen) or Merck (Darmstadt).
  • Cation exchange was performed on a HiTrap SP Sepharose column (Pharmacia) that was equilibrated with buffer A1 (20 mM MES pH 5.5). Cell culture supernatant was diluted 2:1 with buffer A1 and applied to the column (10ml) with a flow rate of 4 ml/min. Unbound sample was washed out with buffer A1 and the bound protein was eluted with 100% buffer B1 (20 mM MES pH 5.5, 1 M NaCI). Eluted protein fractions were pooled for further purification.
  • IMAC was performed, using a HisTrap column (Pharmacia) that was loaded with NiS04 according to the manufacturer's protocol.
  • the column was equilibrated with buffer A2 (20 mM NaPP pH 7.5, 0.4 M NaCI), and the sample was diluted 2:1 with buffer A2 ' to obtain a pH of 7.
  • the sample was applied to the column (2 ml) with a flow rate of 1 ml/min and the column was washed with buffer A2 to remove unbound sample.
  • Bound protein was eluted using a linear gradient of buffer B2 (20 mM NaPP pH 7.5, 0.4 M NaCI, 0.5 M Imidazol) (0-100% buffer B2 in 10 column volumes). Eluted protein fractions were pooled for further purification.
  • Protein concentrations were determined using protein assay dye concentrate (BioRad) and IgG (Biorad) as standard protein. SDS-PAGE under non-reducing conditions was performed with precast 4-12% Bis- Tris gels (NOVEX). Sample preparation and application were according to the manufacturer's protocol. The molecular weight was determined with SeeBlue protein standard (NOVEX). The gel was stained with colloidal Coomassie (NOVEX protocol; Fig 3A) .
  • the purity of the isolated protein was >95% as determined by SDS-PAGE.
  • the molecule had an apparent mass of 45 kDa consistent with the predicted size.
  • the final yield of purified protein was ca. 2.4 mg / 1 cell culture supernatant.
  • the final product ran as an approximately 45 kDa protein under native conditions as determined by gel filtration in PBS. No higher molecular weight forms were detected, suggesting that MOGxCD3 is a monomer (see Fig. 3B).
  • Stably transfected CHO-cells were transferred to 500 ml roller-bottles (Nalge Nunc International) in 250 ml MEM- ⁇ , 500 nM MTX and 5% dialysed FCS. The following day, another volume of medium was added without FCS to obtain a final concentration of 2.5% FCS. Cells were grown for 1 day post confluency. Cells were separated from the supernatant by centrifugation at 4500 rpm, 30 min. in a Rotanta 46 centrifuge, and recombinant protein was purified using a 1-step purification procedure via Protein A affinity chromatography (HiTrap Protein A column, Pharmacia) on the GradiFrac System (Pharmacia).
  • MOG-Fc fusion protein was analyzed via SDS-PAGE as described for MOGxCD3.
  • Coomassie Brilliant Blue staining under reducing conditions revealed a major band at 50kDa, with a higher molecular weight lane at approximately 115kDa.
  • Western blotting under reducing and non-reducing conditions was performed for further analysis (Fig. 4B); under reducing conditions (Fig. 4B, +DTT), MOG-Fc runs as a monomer of 50kDa, which corresponds to its predicted size.
  • the native protein has a molecular mass of approximately 115kDa (Fig. 4B, -DTT) under non-reducing conditions. This suggests that MOG-Fc is a disulfide-bridge linked dimer in its native state, presumably cross-linked via two cysteine residues in the hinge-region of the human lgG1 part of the protein.
  • Example 7 Binding of auto-antigen fusion protein to auto-antibody
  • Hybridoma 8.18-C5 (Linington, MPI Neurobiology Martinsried) was cultivated in serum-free medium (Gibco). Cells were separated from supernatant by centrifugation, and mouse anti-MOG monoclonal lgG1 antibodies were purified using a 1-step purification procedure via Protein G affinity chromatography (HiTrap Protein G column, Pharmacia) on the GradiFrac System (Pharmacia). Column was equilibrated with 10 ml of buffer A (20 mM Tris pH 7.2), and 500 ml of cell culture supernatant were passed through the column. Flow rate was 2 ml/ min. Bound Protein was eluted with 20 mM citrate, pH 3, using a linear gradient. Antibody yield amounted to 4.5 mg/ 1. Protein was > 95% pure as assessed by Coomassie staining.
  • Isolated ⁇ MOG ab 8.18-C5 was . used to detect purified MOG-Fc fusion protein and to verify existence of 1) functional extracellular domain of MOG protein and 2) Fc effector domain in the recombinant protein.
  • MaxiSorp 96-well plates (Nalge Nunc International) were coated with ⁇ MOG at 5 ⁇ g/ ml overnight at 4°C. Plates were blocked with 1% BSA for 1h at RT, washed with PBS/ 0.05% Tween 20.
  • Sandwich-ELISA for detection of M0GxCD3 fusion protein Isolated ⁇ MOG ab 8.18-C5 was used to detect purified MOGxCD3 fusion protein and to verify existence of 1 ) functional extracellular domain of MOG protein and 2) anti-CD3 effector domain in the recombinant protein.
  • MaxiSorp 96-well plates (Nalge Nunc International) were coated with ⁇ MOG at 5 ⁇ g/ ml overnight at 4°C. Plates were blocked with 1% BSA for 1h at RT, washed with PBS/ 0.05% Tween 20.
  • Example 8 Binding of auto-antigen fusion proteins to immune effector cells
  • Buffy coats were diluted 1 :2 in PBS and separated in Ficoll gradient of density 1.077 (Seromed Cat.No. L 6115). Lymphocytes were separated and washed twice with PBS. Erythrocytes were lysed with lysis buffer (8,29g NH4CI cell culture tested (Sigma A-0171 ),
  • MOGxCD3 fusion protein To explore the binding of MOGxCD3 fusion protein to CD3+ cells, 200,000 CD3+ cells were added to each well of a V-bottom microtiter plate (Greiner Laborfechnik). Recombinant MOGxCD3 protein was added to obtain final concentrations of 0.032 up to 100 ⁇ g/ ml in a total volume of 50 ⁇ l per well. Bound fusion protein was detected via binding of 8.18-C5 diluted 1:1000 in FACS-buffer, whereafter bound mouse monoclonal antibody was stained with ⁇ -mouse IgG-FITC (Sigma F-6257) at 1 :40 dilution (Fig. 6).
  • Anti-MOG positive clones were identified, and those showing the greatest amount of MOG-reactivity were expanded and used as targets for in-vitro cytotoxicity assays. More than 90% of cells show cell-surface expression of mlgG1 (Fig. 8A) and bound rMOG (Fig. 8B).
  • a FACS-based cytotoxicity assay was performed. Effector cells (500000), 8.18-C5 target cells and fusion protein were added in a total volume of 200 ⁇ l RPMI/ 10% FCS to each well of a sterile round-bottom multititre plate (CoStar) and incubated overnight at 37°C. Target cells were added to obtain E:T-ratios of 10:1 , and MOGxCD3/-Fc fusion protein was added to attain final concentrations of 0.1 , 1 and 10 ⁇ g/ ml.
  • MOGxCD3 based cytotoxicity was tested by addition of soluble recombinant MOG.
  • a FACS-based cytotoxicity assay was performed as described in example 9, using MOGxCD3 at a concentration of 0.1 ⁇ g/ml.
  • Recombinant, biotinylated MOG (rMOG) was added to a final concentration of 20 ⁇ g/ml and the cytotoxicity assay performed for 16 h. As shown in Fig. 11 , cytotoxic activity can be inhibited by the addition of rMOG.
  • Example 11 MOGxCD3 binds to 8.18-C5 target cells
  • MOGxCD3 Binding of MOGxCD3 to B cell carrying membrane Ig receptor specific for the MOG protein was tested by a FACS-based binding assay. 8.18-C5 target cells were incubated with purified MOGxCD3 fusion protein at various concentrations for 1 h at 4°C. Cells were washed twice with FACS-buffer, and bound fusion protein was detected through its C-terminal HIS-tag using FITC-labeled anti-HIS antibody (Dianova). Cells were analyzed by FACS scanning and mean fluorescence scores were calculated and are shown in Fig. 12. MOGxCD3 efficiently bound at concentrations below 5 ⁇ g/ml.
  • Example 12 Selective elimination of autoreactive B-cells at different effector- to-target ratios (E:T).
  • incubation with MOG-Fc was performed with 10-times the concentration of protein as with splenocytes from anti- MOG transgenic mice (Litzenburger, J. Exp. Med. 188 (1998), 169-180) and for a period of 45 minutes at room temperature compared to 20 minutes on ice.
  • the subsequent incubation with anti-Mad , anti-CD5 or biotinylated 8.18-C5 antibodies was performed on ice for 20 min. As shown in Fig.
  • MOG-Fc did bind to the Mac1 h ' 9h (CD11b, BD Pharmingen) positive population, indicating a preferential binding to macrophages and myeloid CD8 + dendritic cells (DCs).
  • the upper panels of Fig. 14B and C show incubations on ice alone without prior incubation with MOG- Fc at room temperature. No binding of MOG-Fc to CD5 + T cells could be detected emphasizing the selectivity for Fc ⁇ R + cells.
  • the murine monocyte/macrophage cell line p388.D1 also bound MOG-Fc (as described in Example 3 and Fig. 4) as shown by FACS detection of MOG-Fc via FITC-labeled anti-human Fc ⁇ antibody (Fig. 1D).
  • Mean fluorescence intensity (MFI) values observed for MOG-Fc were similar to those obtained with an isotype control lgG1 (Raum, Cancer Immunol Immunother. 50, (2001), 141-150) (data not shown).
  • MOG-Fc The specificity of cell lysis mediated by MOG-Fc (as described in Example 3 and Fig. 4) was investigated by using both unrelated human lgG1 (Raum, Cancer Immunol Immunother. 50, (2001), 141-150) and a mouse B cell line expressing unrelated IgG on its surface (TIB-208).
  • 8.18-C5 target cells were incubated with human PBMCs and 10 ⁇ g/ml MOG-Fc protein (as described in Example 3 and Fig. 4) in RPMI with 10% FCS. Incubation was performed for 16 h at 37°C/5% C0 2 . After incubation, target cells were detected with FITC-labeled anti-murine lgG1 antibody (mlgG, Pharmingen) and analyzed for viability through propidium iodide (PI) staining.
  • FITC-labeled anti-murine lgG1 antibody mlgG, Pharmingen
  • PI propidium iodide
  • Mouse B cell line TIB-208 is expressing cell surface lgG1 of non-MOG specificity and was incubated with human PBMCs and 10 ⁇ g/ml MOG-Fc protein (as described in Example 3 and Fig. 4) in RPMI with 10% FCS. Incubation was performed for 16 h at 37°C and 5% CO z .
  • the mouse B cell line TIB-208 expressing cell-surface IgG of non-MOG specificity was not sensitive to MOG-Fc mediated cell lysis (Fig. 15B).
  • Example 15 MOG-Fc mediated depletion of splenocytes of anti-MOG transgenic mice ex vivo
  • MOG-Fc cytotoxic potential on MOG-specific B cells in vivo
  • the fusion protein was tested in anti-MOG transgenic mice.
  • anti-MOG mice have an extremely high titer of circulating MOG-reactive antibodies (Litzenburger, J. Exp. Med., 188, (1998) 169-180) and secondly, almost all B cells in these mice do show MOG reactivity.
  • MOG-Fc had the ability to deplete primary anti-MOG-positive B cells from anti-MOG transgenic mice ex vivo and in vivo.
  • MOG-reactive B cells Elimination of MOG-reactive B cells ex vivo was efficient (on the order of 70% within 16 hours), while in vivo it was lower but still significant among the population of highly MOG-reactive B cells.
  • MOG-reactive B cells are constantly replenished in the periphery by the bone marrow on the order of 10 r cells/day.
  • the frequency of autoreactive B cells in human autoimmune disease is extremely low (Link, J. Clin. Invest, 87, (1991), 2191-2195 and Nishifuji, J. Invest. Dermatol., 114, (2000), 88-94).
  • the anti-MOG transgenic mouse model represented the most difficult situation and highest possible hurdle to test the in-vivo efficacy of a specific B cell-eliminating protein such as MOG-Fc.
  • MOG-Fc a specific B cell-eliminating protein
  • splenocytes from anti-MOG transgenic mice were isolated and incubated with 10 ⁇ g/ml of MOG-Fc fusion protein for 16 h at 37°C and 5% C0 2 in DMEM with 10% FCS in 5 ml cell culture polypropylene vials (Becton-Dickinson, Pharmingen) at a density of 4x10 6 cells/ml. Lymphocyte analysis was carried out by FACS using biotinylated recombinant MOG protein and antibodies against lgM a , IgD and CD19 (all Becton-Dickinson, Pharmingen).
  • Example 16 In vivo depletion of anti-MOG specific B-cells in transgenic mice
  • the cytotoxic efficacy of MOG-Fc was tested in vivo using the anti-MOG transgenic mouse.
  • This mouse strain carries the anti- MOG heavy chain variable region, derived from the anti-MOG monoclonal antibody 8.18-C5 "knocked in” for the germline JH locus (Litzenburger, J. Exp. Med. 188 (1998), 169-180).
  • FACS FACSCalibur, Becton-Dickinson
  • the percentage of highly MOG expressing B-cells decreased with statistical significance from 45% in control (untreated mice) to less than 30% on day 4 after the first treatment (Fig. 17B).
  • the number of B220 + B cells was decreased to the same extent as the number of IgM-positive B cells (data not shown), suggesting that the reduction of B cells measured by flow cytometry was due to a depletion of cells and not to a down-modulation of the B-cell receptor expression.
  • Example 17 Depletion of autoreactive B cells and reduction of MOG-specific IgG in wildtype mice after cellular transfer
  • Wild-type mice transferred with a limited number of autoreactive B-cells can be used as a model system reflecting the situation in B-cell-mediated autoimmunity which is characterized by the presence of a limited number of autoreactive B-cells versus a high background of endogenous normal B lymphocytes.
  • this system is very close to the real-life situation and confirmes the data on depletion of MOG-reactive B cells obtained in the anti-MOG transgenic mice.
  • Group A was treated with MOG-Fc (100ug each treatment) intraperitoneally 1 , 2 and 3 days post-transfer.
  • Control groups were treated with human lgG1 isotype control (Raum, Cancer Immunol Immunother. 50, (2001 ), 141-150, group B) and PBS (group C).
  • splenocytes derived from anti-MOG transgenic mice with BL/6 background were prepared as single-cell suspension and stimulated with LPS as described (Litzenburger, J. Exp. Med. 188 (1998), 169-180).
  • B cells derived from anti-MOG transgenic mice with BL/6 background were transferred intraveniously into wt BL/6 as described in Example 17.1.
  • Peripheral blood was collected by tail bleeding and sera from day 1 and 3 post-treatment were analyzed for anti-MOG specific IgG titers by ELISA.
  • MOG-Fc had the ability to reduce the number of circulating, MOG-reactive IgG after 24h (Fig. 19A) and 72h (Fig. 19B), presumably via a combination of direct adsorption and neutralization of MOG-reactive immunoglobulin.

Abstract

L'invention concerne une composition destinée à l'élimination sélective des cellules B autoréactives, laquelle comprend au moins une construction de (poly)peptide renfermant au moins deux domaines, un de ces domaines comprenant un antigène autoréactif ou (a) des fragments de celui-ci, reconnus de manière spécifiques par les récepteurs Ig des cellules B autoréactives et un de ces domaines comprenant une molécule effectrice capable d'interagir avec des cellules NK, des cellules T, des macrophages, des monocytes et/ou des granulocytes et/ou d'activer ceux-ci et/ou capable d'activer le système complémentaire.
PCT/EP2001/009714 2000-08-22 2001-08-22 Composition destinee a l'elimination des cellules b autoreactives WO2002016414A2 (fr)

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WO2003068822A2 (fr) * 2002-02-13 2003-08-21 Micromet Ag Constructions (poly)peptidiques desimmunisees
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WO2004031767A3 (fr) * 2002-10-03 2004-06-17 Hospital For Sick Children Res Prevention du syndrome de sjogren primaire par carence de ica69
US7635472B2 (en) 2003-05-31 2009-12-22 Micromet Ag Pharmaceutical compositions comprising bispecific anti-cd3, anti-cd19 antibody constructs for the treatment of b-cell related disorders
WO2011101870A1 (fr) * 2010-02-22 2011-08-25 Transgene Biotek Ltd. Protéines de fusion utilisées pour le traitement de la sclérose en plaques et d'autres maladies auto-immunes
US10662212B2 (en) 2014-03-13 2020-05-26 Universitat Basel Carbohydrate ligands that bind to IGM antibodies against myelin-associated glycoprotein
US11220523B2 (en) 2014-03-13 2022-01-11 Universität Basel Carbohydrate ligands that bind to IgM antibodies against myelin-associated glycoprotein
EP4008725A1 (fr) * 2014-05-02 2022-06-08 The Trustees of the University of Pennsylvania Compositions de récepteurs d'auto-anticorps chimériques et procédés afférents
US10301370B2 (en) 2014-05-02 2019-05-28 The Trustees Of The University Of Pennsylvania Compositions and methods of chimeric autoantibody receptor T cells
EP3166968A4 (fr) * 2014-05-02 2018-05-23 The Trustees Of The University Of Pennsylvania Compositions de récepteurs d'auto-anticorps chimériques et procédés afférents
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US11091591B2 (en) 2015-09-16 2021-08-17 Universität Basel Carbohydrate ligands that bind to antibodies against glycoepitopes of glycosphingolipids
CN110799210A (zh) * 2016-12-02 2020-02-14 得克萨斯A&M大学系统 选择性消耗抗原特异性抗体的融合蛋白
EP3548073A4 (fr) * 2016-12-02 2020-07-22 The Texas A&M University System Protéines de fusion pour l'appauvrissant sélective d'anticorps spécifiques d'un antigène
US11459396B2 (en) 2016-12-02 2022-10-04 The Texas A&M University System Fusion proteins (Seldegs) for selectively depleting antigen-specific antibodies and methods of use thereof

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US20040072749A1 (en) 2004-04-15
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AU2001295503A1 (en) 2002-03-04

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